The role of cilia in recognition of olfactory stimuli has been controversial. Cilia from the intact olfactory rosettes of the rainbow trout Salmo gairdneri were isolated, characterized biochemically, and examined by electron microscopy. The markers studied are those associated with cilia in other organisms. Dynein arms contain Mg2+-AtPase; this enzyme was enriched in the isolated cilia preparation. Guanine nucleotides are associated with the outer microtubule doublets of cilia but adenine nucleotides are not; a substantial enrichment in guanine, relative to adenine, was found in the cilia preparation. Tubulin, the structural protein component of microtubules, occurs in large amounts in cilia. Disc gel electrophoresis indicated tubulin in the cilia preparation. Electron microscopy confirmed the presence of cilia in the isolated preparation. Rainbow trout have an acute sense of smell and many amino acids are odorants to this species. Functional activity of the cilia preparation relevant to odorant recognition was assessed by using binding of radioactively labeled odorant amino acids. L-Alanine, L-serine, L-threonine, L-lysine, and D-alanine bound to the cilia preparation. This study provides direct biochemical evidence that olfactory cilia bind odorant molecules and supports the hypothesis that odorant recognition sites are integral parts of the cilia.